Title: DHFRP1 pseudogene status as a biomarker of chemotherapy response and outcomes in African- American breast cancer patients ABSTRACT African-American (AA) women are diagnosed with more advanced and aggressive breast cancers and have lower survival rates than Caucasian/white women even after controlling for known prognostic factors, treatment differences, and socioeconomic status. Genetic and/or biological factors are thought to play a critical role in disparate survival rates; however, to date few potential biomarkers have been identified to explain AA breast cancer survival disparities and their mechanisms are poorly understood. Our preliminary data suggests that approximately 40% of AA breast cancer patients lack the DHFRP1 pseudogene (DHFRP1-) compared to less than 3% of white/non-AA women, and DHFRP1 status was unrelated to tumor intrinsic subtype. Although DHFRP1- patients initially responded well to neoadjuvant (NA) therapy, they exhibited shorter time to recurrence and worse survival than NA-treated AA or white patients harboring the DHFRP1 pseudogene (DHFRP1+), suggesting that DHFRP1 may play a role in the response to cytotoxic chemotherapy, chemoresistance, and cancer outcomes among AA women. Immortalized lymphoblastoid cell lines (LCLs) derived from DHFRP1- patients inappropriately retained high levels of the dihydrofolate reductase (DHFR) protein upon starvation, suggesting that DHFRP1 may play a functional role in DHFR gene and/or protein regulation. The work herein proposes to expand upon these preliminary findings to determine the clinical relevance of DHFRP1(+/-) status by taking advantage of the unique Lineberger Comprehensive Cancer Center (LCCC) 9830 clinical study at the University of North Carolina at Chapel Hill (UNC) (which is focused on the role of genomic instability and genetic variation in breast cancer risk and therapeutic response) to assess NA response and clinical outcomes (time to recurrence, disease specific survival) in AA patients relative to DHFRP1 status. In vitro studies will also examine patient-derived LCLs and AA breast cancer cell lines to characterize the genetic and molecular role(s) of DHFRP1 in chemosensitivity and resistance. We propose a novel mechanism mediated by DHFRP1 status by which excessive DHFR protein levels results in a hyper-mutagenic cellular microenvironment that predisposes these cancer cells to high levels of genomic instability leading to cytotoxic chemotherapy resistance. The results obtained upon completion of these studies are expected to lay the groundwork for alternative personalized therapeutic strategies for these high-risk AA breast cancer patients.